Development 129, 945-955 (2002) 945 Printed in Great Britain © The Company of Biologists Limited 2002 DEV2828

Distinct functions of TBP and TLF/TRF2 during : requirement of TLF for heterochromatic chromocenter formation in haploid round

Igor Martianov1,†, Stefano Brancorsini1,†, Anne Gansmuller1, Martti Parvinen2, Irwin Davidson1,‡ and Paolo Sassone-Corsi1,‡ 1Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, B.P. 163, 67404 Illkirch Cédex, C.U. de Strasbourg, France 2Department of Anatomy, University of Turku, 20520 Turku, Finland †These authors contributed equally to this work ‡Authors for correspondence

Accepted 23 November 2001

SUMMARY

TLF (TBP-like factor) is a protein commonly thought to pachytene , suggesting a function prior to the belong to the general transcription initiation complex. TLF apoptosis of the haploid cells. A refined study of TLF- is evolutionarily conserved and has been shown to be deficient mice reveals defective formation in early essential for early development in C. elegans, zebrafish and stage spermatids. Most importantly, our results uncover an Xenopus. In mammals however, TLF has a specialised unsuspected function of TLF in organisation. function, as revealed by targeted mutation of the gene in Indeed, early spermatids in TLF-deficient mice display a the mouse germline. The TLF mutation elicits a complete fragmentation of the chromocenter, a condensed structure arrest of late spermiogenesis and increased haploid cell formed by the association of centromeric heterochromatin apoptosis. We explored in more detail the molecular and containing the HP1 proteins. This defect is likely to be function that TLF plays in the differentiation program of the primary cause of spermatogenic failure in the TLF male germ cells. A comparison of TBP and TLF reveals mutant mice. drastic differences, both in their temporal expression pattern and in their intracellular location. While TBP is ubiquitously expressed, TLF expression is strictly Key words: Spermatogenesis, TLF, TBP, Chromatin, Chromocenter, developmentally regulated, being very high in late HP1, Mouse. Rat

INTRODUCTION Burley, 1994; Kim et al., 1993) which is responsible for DNA binding, and interaction with TFIIA, TFIIB and TAFs (Liu et RNA polymerase II transcription initiation in eukaryotes al., 1998; Nikolov et al., 1995; Tan et al., 1996). Two proteins requires the formation of a multiprotein complex around the with strong sequence similarity to the core domain of TBP have mRNA start site. (Hampsey, 1998; Hampsey and Reinberg, been described. The first, Drosophila melanogaster TRF1, is 1999; Orphanides et al., 1996). A central factor in this process expressed in a tissue-specific fashion and functions as both a is transcription factor TFIID comprising the TATA binding RNA polymerase II and polymerase III transcription factor protein (TBP) and 14 additional TBP-associated factors (Hansen et al., 1997; Holmes and Tjian, 2000; Takada et al., (TAFIIs). TFIID plays an important role in transcriptional 2000). At present the gene for this has been described only in regulation through contributing to promoter recognition and Drosophila. acting as a target for transcriptional activators (for reviews, see A second protein with high homology to the TBP core Albright and Tjian, 2000; Bell and Tora, 1999; Gangloff et al., domain has received various names; TLF, TRF2, TLP and TRP 2001; Green, 2000). TBP itself is thought to play a role in (Maldonado, 1999; Moore et al., 1999; Ohbayashi et al., 1999; transcription initiation by all three RNA polymerases through Rabenstein et al., 1999; Teichmann et al., 1999; Wieczorek et its presence in TFIID for polymerase II, SL1 for polymerase I al., 1998). The TLF (TBP-like factor) gene is found in most and TFIIIB for polymerase III (Chiang et al., 1993; Comai et metazoans (Dantonel et al., 1999) and multiple sequence al., 1992; Hernandez, 1993). alignments show that TLFs contain two direct repeats, which TBP has a bipartite structure with a variable N-terminal are well conserved between all TBP-type factors. Despite this domain and a highly conserved C-terminal core domain. The sequence similarity, TLF does not bind classical TATA core domain of TBP folds into a saddle-like structure (Kim and elements (Dantonel et al., 1999; Moore et al., 1999), but 946 I. Martianov and others does interact with TFIIA and TFIIB. Transfection, and while in TLF–/– spermatids the single center is fragmented into overexpression studies in cells and in in vitro experiments, multiple structures. These results show that TLF plays an have shown that TLF associates stably with TFIIA and can act essential role in proper chromatin organisation in developing as a repressor or activator of RNA polymerase II transcription male germ cells. Disorganisation of chromatin domains in the (Moore et al., 1999; Ohbayashi et al., 2001; Teichmann et al., early round spermatids could lead to defects in gene expression 1999). and chromosomal packaging in the elongated spermatids and The physiological function of TLF was first addressed using therefore be the primary cause of differentiation arrest and RNAi in Caenorhabditis elegans where TLF appears to apoptosis of TLF–/– spermatids. function as an essential RNA polymerase II transcription factor (Dantonel et al., 2000; Kaltenbach et al., 2000). Injected embryos die at an early stage of embryogenesis before the MATERIALS AND METHODS onset of morphogenesis. Similar results have been obtained using antisense strategies in Xenopus laevis (Veenstra et al., RNA analysis 2000) and in the zebrafish Danio rerio (Muller et al., 2001). Total RNA was extracted from mouse testis and rat spermatogenic These studies underscored a critical role of TLF in the cells as previously described (Chomczynski et al., 1986) and analyzed embryogenesis of these species. It has been suggested that TLF by RNase protection (Foulkes et al., 1991). Rat and may act as a surrogate for TBP at specific promoters whose cells were separated by centrifugation as described (Meistrich et al., 1981). The TLF and TBP cDNAs were used to expression is required at the onset of zygotic transcription in [α 32 ] the developing embryos. However, as it has been demonstrated generate a - P UTP antisense riboprobe using an in vitro transcription kit (Promega). CREM riboprobe was as described recently, the situation is radically different in mammals. previously (Molina et al., 1993). In all RNase protection analyses, Indeed, using homologous recombination, it has been shown transfer RNA was used as a control for nonspecific protection. A that mouse TLF is not required for embryogenesis, but that it mouse β-actin riboprobe was used as internal control to monitor the is essential for spermatogenesis (Martianov et al., 2001; Zhang loading of equal amounts of RNA. et al., 2001b). Spermatogenesis is a cyclic process in which diploid Immunoblots and immunohistochemistry spermatogonia differentiate into mature haploid spermatozoa. Rat segments at defined stages were isolated Spermatogonia committed for differentiation give rise to using the transillumination-assisted microdissection method spermatocytes which undergo two meiotic divisions, to (Parvinen and Hecht, 1981) and pooled to obtain 4-5 cm, equivalent generate haploid round spermatids. During the process of to 4-5 mg of wet weight of tissue. Protein extracts were made by shearing the tubule segments in 2× boiling Laemmli buffer containing spermiogenesis the haploid round spermatids undergo an 10 mM β-mercaptoethanol. The extracts were analyzed by SDS- elongation phase in which they are sculptured into the shape PAGE and staining with Coomassie Blue stain to normalise each of mature spermatozoa. This entails a major biochemical and preparation. Immunoblotting and chemiluminescence detection were morphological restructuring of the germ cell in which the performed by standard methods. Immunohistochemistry was majority of the somatic histones are replaced by protamines to performed on fixed sectioned seminiferous tubules or from squash pack the DNA into the sperm cell nucleus. In the absence of preparations of microdissected tubules from wild-type or TLF mutant TLF, round step 7 haploid spermatids die through apoptosis as mice as previously described (Martianov et al., 2001; Nantel et al., the transition to elongated spermatids begins. Rare elongating 1996). Antibodies against TBP, TLF and CREM are as previously described (Brou et al., 1993; Delmas et al., 1993; Martianov et al., spermatids remain beyond this stage but rapidly degenerate α such that no spermatids survive beyond step 13. Hence, TLF 2001). The anti-HP1 , 2HP1-1H5 and anti-fibrillarin antibodies are also as described previously (Fomproix et al., 1998; Nielsen et al., is essential for the differentiation of male germ cells. These 2001). results highlighted the non-redundant function of TBP and TLF in mammals and solicited further investigations aimed at identifying their distinct roles. To gain further insight into the molecular function that TLF RESULTS plays in the differentiation program of male germ cells, we have examined in detail the pattern of TLF expression during Developmental and stage-specific expression of TLF spermatogenesis and compared it with that of TBP. Our results and TBP show marked differences in temporal regulation of TBP and To compare the functions of TBP and TLF in developing male TLF as well as differences in their intracellular location. In germ cells, we first analysed their respective expression particular, TLF is strongly expressed in late pachytene patterns. During the first wave of spermatogenesis, germ cells spermatocytes, suggesting that it has a function prior to the are synchronised in their development, different cell types apoptosis of the haploid cells. A detailed analysis of the germ appearing at different times. The developmental onset of TBP cell phenotype in TLF–/– (Tlp–/–) mice shows that is indeed the and TLF expression was examined by RNase protection case since defective acrosome formation is observed in early analysis with 1- to 4-week-old mouse testes. stage spermatids. Our results also reveal an unexpected role Low expression of TBP and TLF was observed in 1- to 2- of TLF in chromatin organisation. Strikingly, early stage week-old immature testes (Fig. 1A, lanes 2-3). At three weeks, spermatids of TLF-deficient mice are characterised by a strong increase in expression of both TBP and TLF was fragmentation of the chromocenter, a condensed structure observed, reaching levels similar to those observed at 4 weeks formed by the association of centromeric heterochromatin and in the adult (lanes 3-5). The increase in expression (Hoyer-Fender et al., 2000; Brinkley et al., 1986; Zalensky et correlates with the accumulation of late pachytene al., 1993). Wild-type spermatids contain a single chromocenter, spermatocytes and round spermatids, which occurs at this time. TLF in spermiogenesis 947

Fig. 1. Developmental regulation of TBP and TLF. (A) RNase Fig. 2. (A,B) Expression of TLF and TBP in seminiferous tubules of protection experiments. The RNA used is indicated above each lane wild-type (A) and TLF null-mice (B). The upper panel in each and the detected transcripts identified are listed to the right of each section shows the immunodetection of TLF and TBP as indicated. panel. Lane 1 shows a control with only tRNA. (B) The origin of the The middle and lower panels show the corresponding Hoechst × RNA, pachytene spermatocytes (spermatocytes), and haploid stained DNA and merged images. 40 magnification. spermatids (spermatids) is shown above each lane and the transcripts are listed to the right of each panel. (C) Immunodetection of TBP, and TLF overexpression precedes that of the haploid cell- TLF and CREM. The stage-specific protein extracts used are specific CREM activator (Fimia et al., 2001) (Fig. 1A). Hence, indicated above each lane and the locations of TBP, TLF and the CREM isoforms are indicated to the right of each panel. TLF is overexpressed in both late meiotic and post-meiotic cells. In a more refined analysis, TBP and TLF expression were This observation is consistent with previous reports of TBP examined in purified adult rat pachytene spermatocytes and overexpression in meiotic and post-meiotic cells (Persengiev et haploid spermatids. TBP and TLF mRNAs strongly al., 1996; Schmidt and Schibler, 1995) with the fact that TBP accumulate in pachytene spermatocytes, while lower levels are 948 I. Martianov and others

Fig. 3. (A-D) Immunodetection of TBP and TLF in developing male germ cells. TLF and TBP are detected in microdissected segments of seminiferous tubules from various stages as indicated above each panel. Representative examples of cell types are indicated. PS, pachytene spermatocytes; RS, haploid round spermatids; ES, elongating spermatids; EES, early elongating spermatids; LS, leptotene spermatocytes; ZS, zygotene spermatocytes. 40× magnification.

western blotting using specific monoclonal antibodies (Brou et al., 1993; Lescure et al., 1994; Martianov et al., 2001). TBP was detected at each stage with highest expression seen at stage VII (Fig. 1C, lanes 5-6). In contrast, TLF accumulation was weak at stage I, but increased through stages II-VI to peak at stage VII before again decreasing through stages VIII- XIV (Fig. 1C). As a control, strong expression of the CREMτ, τ1 and τ2 isoforms was observed from stages IV- VIII (Fig. 1C, lanes 3-7). These results show that TBP and TLF have a similar developmental onset of expression, but that they are differentially expressed at different stages of the spermatogenic cycle. Expression of TLF is dynamic and cell specific, while TBP is ubiquitous The monoclonal antibodies against TBP and TLF were used to examine the expression and localisation of the corresponding proteins at different stages of differentiation, by immunohistochemistry. We first compared their expression profiles in sectioned seminiferous tubules from wild-type mice. TLF expression is cell- specific and is not visible in any of the randomly sectioned tubuli, indicating that it is dynamically regulated during the cycle (Fig. 2A). In contrast, expression of TBP is much more present in haploid spermatids (Fig. 1B, compare lanes 2 and homogeneous as it is present at all stages in all cell types with 3). The converse was observed for the CREM activator mRNA the exception of the more mature elongating spermatids (Fig. (Fig. 1B) (Delmas et al., 1993). 2A). We also examined TBP expression in the seminiferous Spermatogenesis occurs in synchronised waves within the tubules from TLF–/– mice to investigate whether its expression seminiferous epithelia. The cycle is divided into 12 stages in was altered in the absence of TLF. As expected, no TLF mouse and 14 stages in rat, where each stage comprises a mix expression could be seen in the tubuli from the mutant animals. of cells in given developmental steps. We isolated individual TBP was however expressed in all remaining cell types similar segments from rat seminiferous tubuli corresponding to to that observed in wild-type tubuli (Fig. 2B). each developmental stage by transillumination-assisted To examine the expression of TBP and TLF in more detail, we microdissection (Parvinen and Hecht, 1981) and examined isolated segments of mouse seminiferous tubuli corresponding the accumulation of TBP and TLF proteins at each stage by to each developmental stage by transillumination-assisted TLF in spermiogenesis 949

Fig. 4. (A-C) Immunodetection of TBP and TLF in developing male germ cells. The layout and abbreviations are as described in Fig. 3. microdissection and subjected them to A TLF expression immunostaining with the monoclonal antibodies against TLF and TBP. MS At stage V, nuclear and cytoplasmic expression of TLF in pachytene spermatocytes was observed. TLF was also expressed in step 5 haploid round spermatids, predominantly in the nucleus (Fig. 3A). In contrast, no TLF expression was observed in the RS ES maturing elongated spermatids at this stage. TBP P P D was strongly expressed in the nucleus of both the Z pachytene spermatocytes and the round spermatids, PL L but like TLF no expression was seen in the SG elongated spermatids. At stage VII, a similar, but not identical situation is observed. Expression of TLF is now predominantly, but not exclusively, nuclear in both the pachytene spermatocytes and B TBP expression step 7 round spermatids (Fig. 3B). Strong nuclear expression of TBP is also seen in these cell types at MS this stage. Interestingly, although no expression of TLF is seen in the now almost mature step 16 , perinuclear expression of TBP can be seen (see also below). RS ES At stage VIII-IX, TLF expression persists in the P P D pachytene spermatocytes, but only very weak expression in the step 9 early elongating spermatids PL L Z is observed (Fig. 3C). In contrast, no TLF SG expression is observed in leptotene spermatocytes.

Fig. 5. (A,B) Summary of TLF and TBP expression Stages of the cycle during spermatogenesis. The spermatogenic wave is PL=preleptotene Z=zygotene RS= round spermatids schematised and the representative cell types are SG=spermatogonia D=diakinetic ES=elongating spermatids L=leptotene P=pachytene indicated. Expression of TLF is indicated in A by the red MS= mature spermatozoa Stem cells Primary spermatocytes Secondary spermatocytes Haploid cells colouring and that of TBP in B by the green colouring. 1st meiosis 2nd meiosis 950 I. Martianov and others

TBP is expressed in the leptotene spermatocytes, but its expression is much stronger in the pachytene spermatocytes, while it strongly declines in the early elongating spermatids. By stage X expression of TLF in the pachytene spermatocytes has become exclusively nuclear and only very weak expression is observed in the elongating spermatids (Fig. 3D). No TLF expression is seen in the zygotene spermatocytes. TBP expression continues to decline in the elongating spermatids, while it remains strong in the pachytene spermatocytes and weaker in the zygotene spermatocytes. Strikingly, as the elongating spermatids mature, a strong nuclear/perinuclear expression of TLF is observed beginning at stage XI and becoming stronger at stage XII (Fig. 4A,B). TLF expression persists in step 13 and 14 elongating spermatids and becomes cytoplasmic (Fig. 4C,D). In contrast, no TBP is expressed in the elongating spermatids at this time. TBP is however strongly expressed in the early step 1-2 haploid round spermatids and in the pachytene spermatocytes. Little or no expression of TLF is seen in these cell types at this stage. The above results indicate that TLF is expressed at three stages in the spermatogenic wave: in stage IV-XI pachytene spermatocyes where its expression is at first predominantly cytoplasmic, but later becomes nuclear, in stage IV-VII haploid spermatids, and in step 11-15 elongating spermatids where its expression is perinuclear and cytoplasmic (summarised in Fig. 5A). In between these stages, TLF expression is weak or undetectable. In contrast, TBP is expressed in all spermatocytes, although its expression strongly increases in late pachytene spermatocytes. TBP is strongly expressed at all stages in round haploid spermatids and its expression declines rapidly at the transition to elongating spermatids. Finally, TBP expression is observed in late step 16 elongating spermatids (summarised in Fig. 5B). Confocal microscopy was used to compare the intracellular localisation of TLF and TBP. Perinuclear and cytoplasmic TLF expression was observed in elongating spermatids (Fig. 6A, top panel). In contrast, no TBP expression was detected in elongating spermatids (Fig. 6A, middle panel), but perinuclear expression is however seen in late step 16 elongating spermatids as described above (Fig. 6A, lower panel). In round spermatids, TLF was expressed homogeneously in the nucleus, including the chromocenter, whereas most TBP was clearly excluded from the chromocenter (Fig. 6A, compare upper and middle panels). In pachytene spermatocytes, TLF was found in both the and nucleus, whereas TBP was nuclear Fig. 6. Different intracellular localisations of TBP and TLF. (Fig. 6B). Within the nucleus, TBP was excluded from the (A) Confocal images of sectioned seminiferous tubules stained with condensed heterochromatin, while TLF was present in both the antibodies against TLF (upper panels) or TBP (lower panels) as heterochromatic and euchromatic compartments. Exclusion of indicated. A single representative section is shown. RS, haploid TBP from the heterochromatin compartment of pachytene round spermatids; ES, elongating spermatids; C, the chromocenter. The lower panel shows perinuclear TBP detected in late stage spermatocytes and haploid spermatids is not easily seen in elongated spermatids. (B) Confocal sections of pachytene standard immunofluorescence images as presented in Figs. 3 spermatocytes (PS) stained with TLF or TBP as indicated. In all and 4 which do not result from a single focal plane, but rather panels the middle and right hand images show the corresponding from the nuclei as whole. The confocal images from a single Hoechst stained DNA and merged images respectively. 400× focal plane give a more accurate picture of TBP distribution. magnification. These results reveal significant differences in the intranuclear distribution of TBP and TLF in both pachytene spermatocytes and round spermatids. in germ cell differentiation before apoptosis is observed in step 7 spermatids. To assess this possibility a comprehensive Histological analysis of germ cells in TLF–/– histological analysis has been performed using seminiferous tubuli transillumination-assisted microdissected segments. In The TLF expression profile suggests that it may have a function segments from TLF–/– mice, no mature spermatozoa were TLF in spermiogenesis 951

Fig. 7. Live germ cell cytology in microdissected segments of seminiferous tubules from wild-type (WT) and TLF–/– (–/–) mice. (A) Stage VII segment from a wild-type mouse. (B,C) Stage VII segments from a TLF–/– mouse. (D) Stage VI segment from a TLF–/– mouse. EA and LA, early and late apoptotic round spermatids respectively; C, chromocenter; S, . A, acrosome. Ap, apoptotic round spermatids; D degenerating round spermatids. ES16. Step 16 elongated spermatid. 1000× magnification. observed, but extensive apoptosis of stage 7 round spermatids vacuolated and/or not properly associated with the head (Fig. was evident. Numerous early and late apoptotic round 8G,I compared with wild type, F,H). Hence, TLF deficiency spermatids are seen in segments from TLF–/– mice (Fig. 7B), causes acrosomal abnormalities in round spermatids before the whereas no such cells are observed in wild-type segments (Fig. onset of apoptosis. 7A). Extensive phagocytosis of apoptotic round spermatids by Sertoli cells was observed (Fig. 7C). In contrast, pachytene Absence of chromocenter formation in TLF–/– round spermatocytes appeared normal. No apoptotic round spermatids spermatids were observed at earlier stages, but rare Histological analysis of round spermatids showed that the degenerating elongating spermatids were observed (Fig. 7D). chromocenter, visible as a single dark body in normal These observations are consistent with those previously spermatids, was fragmented in TLF mutant spermatids (see described (Martianov et al., 2001; Zhang et al., 2001b). C in Fig. 7A,D). To avoid possible confusion between the chromocenter and the nucleolus, we validated the location of Abnormal acrosome formation in TLF–/– round the nucleolus by performing immunofluorescence with an anti- spermatids fibrillarin antibody, which selectively identifies the nucleolus. Acrosome maturation begins in stage I-II round spermatids and In early stage I-II spermatids, the anti-fibrillarin antibody can be divided in three stages: Golgi, cap, and acrosomal (for reveals the presence of 2-3 small nucleoli per nucleus, while a review, see Abou-Haila and Tulsiani, 2000). During the staining of DNA with Hoechst revealed the presence of a single Golgi-phase, small proacrosomal granules appear from the dense staining chromocenter in the middle of the nucleus, as and coalesce to form the acrosomal granule previously described (Fig. 9A) (Brinkley et al., 1986; Hoyer- the membrane of which fuses with the outer membrane of the Fender et al., 2000; Zalensky et al., 1993). The merged image nuclear envelope. Newly synthesised granules and cisternae substantiates that the nucleoli and the chromocenter are distinct of the Golgi then form the acrosomal vesicle around the structures in spermatids. While the nucleolar staining in the acrosomal granule. The cap-phase corresponds to the TLF–/– spermatids was comparable to wild type, the Hoechst expansion of the vesicle over the anterior half of the nucleus. staining clearly shows that the chromocenter is incompletly In the acrosomal-phase, the contents of the acrosomal condensed and fragmented. Instead of one single center, three vescicule are gradually distributed throughout the cap to form or more smaller structures are visible. This result is confirmed the final acrosome. Although no increase in apoptosis was by electron microscopic examination. In wild-type spermatids, observed in early stage round spermatids of the TLF-deficient a single chromatin condensation in the center of the nucleus is mice, we have found defects in acrosome formation. observed along with a very small and dense nucleolus as Histological analysis revealed that the acrosome was observed in the fibrillarin staining (Fig. 8J). In mutant asymmetric in TLF–/– spermatids since the darker acrosomic spermatids, several less dense and more peripheral granule, normally situated in the middle, was found at the edge condensations are observed analogous to that observed with of the acrosome system (Fig. 7D). Electron microscopy Hoechst staining (Fig. 8K). indicated that the acrosomal granules did not coalesce in stage It has previously been shown that the heterochromatin II-III spermatids of TLF–/– mice, but were found in the protein M31 (HP1β), which is associated with constitutive cytoplasm in an asymmetric position, on the outer membrane heterochromatin in somatic cells (Minc et al., 1999), localises of the nucleus, or invaginated in the nucleus (Fig. 8B-E to the chromocenter in spermatids (Hoyer-Fender et al., 2000). compared with wild-type Fig. 8A). In the rare surviving To determine whether the closely related HP1α protein is also elongating spermatids at stages IX-XI, were localised to the chromocenter in spermatids and whether 952 I. Martianov and others

uniquely associated with the chromocenter in wild-type spermatids (Fig. 9B). In mutant spermatids, HP1α colocalises with each of the fragmented heterochromatic foci (Fig. 9B). In contrast to spermatids, where abnormal heterochromatin localisation is seen, the distribution of heterochromatic loci as observed by Hoechst staining and HP1α localisation was comparable in both wild-type and mutant pachytene spermatocytes (Fig. 9B). These results indicate that defects in heterochromatin organisation occur only in the round spermatids. Therefore, the organisation of heterochromatin is dramatically altered in TLF–/– spermatids, underscoring a critical role for TLF in this process.

DISCUSSION

The identification of TBP-like factors posed a number of questions on their function, their expression patterns, and their molecular mechanisms of action. The finding that mammalian TLF is not essential for embryonic development, as its counterparts in other organisms (Dantonel et al., 2000; Kaltenbach et al., 2000; Muller et al., 2001; Veenstra et al., 2000), but is instead crucial for the differentiation program of spermiogenesis (Martianov et al., 2001; Zhang et al., 2001b), begs the question of what are the distinct functions of TLF and TBP in this system. We have explored this issue by comparing the detailed expression patterns of TBP and TLF in male germ cells. This approach has allowed the uncovering of an unexpected function of TLF in the heterochromatic organisation of the chromocenters of round spermatids. Distinct expression patterns of TLF and TBP in male germ cells Expression of TBP and TLF genes is strongly upregulated with the appearance of pachytene spermatocytes and haploid spermatids at post-natal week 3-4, slightly earlier than the upregulation of the transcriptional activator CREM, which is restricted to haploid cells (Foulkes et al., 1992). High expression of TBP and TLF in late pachytene spermatocytes is confimed by RNase protection using purified pacyhtene cells. Our present results are in agreement with those previously obtained showing a developmental upregulation of TBP and TLF expression (Schmidt and Schibler, 1995; Zhang et al., 2001a). However, while it has been shown that TLF mRNA is Fig. 8. Electron microscopic sections of wild-type and TLF–/– expressed in pachytene and round spermatids (Martianov et al., seminiferous tubules. (A-I) High magnification views of defective 2001; Zhang et al., 2001a), it has previously been suggested acrosome formation. (J) Wild-type and (K) mutant cell showing that TBP mRNA upregulation and overexpression was specific position of . (A) Normal acrosome in wild-type spermatids; (B-E) defective acrosome in round spermatids; to round spermatids (Schmidt and Schibler, 1995). The results (F-G) acrosome in early wild-type or mutant elongating spermatids; on mRNA expression and immunofluorescence (see below) (H-I) defective acrosome in late step 13 wild-type or mutant shown here indicate that TBP is also strongly overexpressed in elongating spermatids. AG, acrosomal granule; G, Golgi; late pachytene spermatocytes and is not restricted to haploid N, nucleolus; V, vacuole; C, chromocenter; A, acrosome. B shows a cells (see also Persengiev et al., 1996). close-up of the acrosome of the cell shown in K. A,C-G: 4000× The differences in TBP and TLF expression are highlighted magnification; B,H-K, 5000× magnification. when the respective proteins are detected by immunofluorescence. TBP is present in all cell types, although it is expressed at much higher levels in pachytene fragmentation of the chromocenter observed here in the spermatocytes and haploid round spermatids compared to Hoechst staining is accompanied by a mislocalisation of HP1α, zygotene and leptotene spermatocytes. This is in agreement we performed immunofluorescence with an anti-HP1α with the high mRNA expression seen in the pachytene monoclonal antibody (Nielsen et al., 2001). As described for spermatocytes and confirms that TBP overexpression begins in M31(HP1β) (Hoyer-Fender et al., 2000), HP1α is also late meiotic cells. TBP levels decrease as the spermatids TLF in spermiogenesis 953

Fig. 9. Immunodetection of fibrillarin and HP1α in wild-type and TLF mutant spermatids. (A) Immunodetection of fibrillarin in wild-type (above) and mutant (below) spermatids. Representative nucleoli (N) and chromocenters (C) are indicated. (B) Immunodetection of HP1α. Nomenclature is as described in A.

By step 10 little TLF is visible, but it reappears in a perinuclear and eventually cytoplasmic location at steps 12-14. This expression pattern suggests that TLF functions at multiple steps in the differentiation pathway and not only in step 7 spermatids when apoptosis takes place. The results of the histological analysis presented here provide evidence that this is indeed the case. We observe severe acrosomal defects in early stage spermatids. These defects include aberrant localisation and lack of coalescence of the acrosomal granules and vacuolisation. Despite these abnormalities, we did not observe defects in expression of genes involved in acrosome formation, such as proacrosin binding protein (Martianov et al., 2001), Tpx-1 and Sp56 (data not shown). A more thorough analysis of TLF–/– spermatids will be required to identify the changes in gene expression responsible for these defects. TLF is essential for heterochromatin organisation in haploid cells In normal mammalian spermatids, the centromeric heterochromatin of elongate, but surprisingly, perinuclear TBP can be detected each chromosome is condensed into a single chromocenter again in almost mature spermatozoon. This late expression structure (Brinkley et al., 1986; Zalensky et al., 1993). This may be due to the translation of stored mRNA, as is often the structure contains the known heterochromatin associated case for transcripts in germ cells (for a review, see Steger, protein M31 (HP1β) (Hoyer-Fender et al., 2000) and was found 1999). Alternatively, it is possible that TBP molecules are to serve as an organiser for the subsequent packaging of DNA present at all stages during the elongation phase, but are in a well defined chromosomal topology in the sperm cell shielded from the antibody by the condensed chromatin. nucleus (Meyer-Ficca et al., 1998; Zalensky et al., 1995). We TLF presents a more dynamic pattern of expression. It is first show here that the structuring of the chromocenter is disrupted present in stage IV-V pachytene spermatocytes, where it is both in TLF–/– spermatids. Instead of a single center, multiple cytoplasmic and nuclear, before becoming restricted to the smaller structures are observed. The heterochromatin- nucleus at later stages. This result differs from that reported by associated protein HP1α is localised uniquely at the Zhang et al. (Zhang et al., 2001a) where TLF mRNA was first chromocenter in wild-type spermatids and is found in each of detected only in stage VIII pachytene spermatocytes. The onset the smaller bodies in mutant spermatids, confirming that they of expression is clearly prior to this stage. TLF expression correspond to heterochromatic foci. These results demonstrate decreases sharply in early round spermatids, but reappears in that TLF is essential for the formation of the chromocenter and stage IV spermatids and persists during the early elongation steps. thus for heterochromatin organisation in spermatids. 954 I. Martianov and others

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